Regenerative medicine represents an exciting new approach in the treatment of human disease. Progress has been made in strategies for the regeneration of a variety of tissues including skin, corneal epithelium, cartilage, bone and bladder. Tissue regeneration would represent an important advancement in the treatment of salivary gland hypofunction that occurs in the autoimmune disease Sjogren's syndrome and as a result of 3-irradiation therapies for head and neck cancers. To develop techniques for the successful regeneration of salivary glands in vivo, it is first necessary to understand signaling pathways that regulate gland development, which involves acinar formation (i.e., cell proliferation and migration) and organization of acini into a functional gland structure (i.e., cell differentiation into a polarized epithelium with high transepithelial resistance). Towards the goal of regenerating salivary gland tissue, our studies have identified a P2Y2 nucleotide receptor (P2Y2R) that is upregulated in diseased or damaged salivary glands. These published and preliminary findings have demonstrated that P2Y2R activation regulates signaling pathways that mediate cell proliferation and migration and the maintenance of tight junctions required for normal saliva secretion. Over the past 15 years, we have identified novel structural motifs in the P2Y2R that facilitate interactions with complex signaling networks known to regulate cell growth and differentiation involving integrins, growth factor receptors, matrix metalloproteases, adhesion molecules and cytoskeletal proteins. These signaling networks enable the P2Y2R to regulate cellular responses, including migration and proliferation, integrin/extracellular matrix interactions, cytoskeletal rearrangements and the expression of cell adhesion molecules and tight junction proteins. Collectively, these results strongly support the hypothesis that expression and activation of P2Y2Rs in damaged or diseased salivary gland epithelium promote tissue repair and regeneration. Accordingly, we propose to utilize in vitro models of acinar formation to demonstrate that activation of P2Y2Rs enhances regenerative responses. We also will use in vivo submandibular gland (SMG) duct-ligation to validate a role for P2Y2Rs in the recovery and regeneration of damaged salivary glands. We have organized these hypotheses around several Specific Aims. Specific Aim 1 will identify P2Y2R signaling pathways that mediate acinar formation. Specific Aim 2 will investigate the role of integrin/P2Y2R interactions in cellular responses required for acinar formation. Specific Aim 3 will determine whether P2Y2R interaction with the actin cytoskeleton regulates tight junction organization in Par-C10 monolayers. The 3 Specific Aims in this proposal are designed to identify the mechanisms whereby P2Y2R upregulation and activation enhances salivary gland regeneration towards developing better therapies to repair damaged salivary glands. PUBLIC HEALTH RELEVANCE: Salivary glands are damaged by diseases such as Sjogren's syndrome and as a result of 3-irradiation therapies for head and neck cancers. Our studies have identified a receptor on the cell surface, the P2Y2 nucleotide receptor (P2Y2R) that is upregulated in diseased or damaged salivary glands. Previous studies have demonstrated that P2Y2R activation regulates signaling pathways that mediate cell proliferation, migration and differentiation, which strongly support the hypothesis that expression and activation of P2Y2Rs in damaged or diseased salivary glands can be used to promote tissue repair and regeneration.